Percutaneous interventions have become the primary therapy for many athero-occlusive lesions, but suffer from a 30 percent restenosis rate due to the development of intimal hyperplasia, a lesion that results from the abnormal migration and proliferation of vascular smooth muscle cells. Smooth muscle cell migration in response to uPA depends on its binding to uPAR, a unique cell surface receptor which is pertussis toxin sensitive and whose signaling involves G-protein activation. The goal of this proposal is to define the uPAR pathway with particular reference to uPAR-G-protein interactions and to identify the downstream elements involved. The central hypothesis we plan to test is that uPAR activation of the heterotrimeric G-proteins leads to activation of the small G-proteins ras, rac and rho that in turn induces ERK-dependent, cell migration. Specifically, this proposal will: 1) Define the contribution of G-protein signaling in uPAR mediated migration: We will characterize the migratory response to sc-uPA, N terminal fragment of uPA and C-terminal fragment of uPA in presence and absence of G(xi and Gpy inhibitors. We will examine the time course of ERK activation, the impact of Goci and Gpy inhibitors, and will identify the Gui/oy subunits associated with the receptor. 2) Characterize the contribution of specific Goti and Goy to uPAR signaling: We will transfect cells with constructs for appropriate active and inactive mutants of Goti and Goy, and examine the effects of alterations of these pathways on migration in response to the N-terminal fragment of uPA. Parallel experiments using uPA-/- and uPAR -/- cells will be used to determine the need for the presence of uPA and uPAR. 3) Elucidate the role of small G-protein pathways in uPAR signal transduction: Migration involves the activation of a cascade of several small G-proteins, which can be activated by the heterotrimeric G-proteins G(x and Gpy. We will quantitate ras activity in the membrane and examine the migratory response of the cells to uPA in cells, we have transfected with active or inactive mutants of ras and rac. Ras can activate P13Kinase and we will define the role of ras activated raf and ras-induced P13Kinase activation of raf on uPAR induced migration. Understanding the mechanisms and control of cell migration in vascular lesions is essential in advancing our knowledge of the pathophysiology of vessel remodeling. It is anticipated that data generated from these studies will provide valuable insights for the development of therapeutic intervention to address clinical relevant problems of vessel restenosis. (End of Abstract)